Infantile globoid cell leucodystrophy (GLD, galactosylceramide lipidosis or Krabbe's disease) is a rare, autosomal recessive hereditary degenerative disorder in the central and peripheral nervous systems. The incidence in the US is estimated to 1:100.000. It is characterised by the presence of globoid cells (cells with multiple nuclei), degeneration of the protective myelin layer of the nerves and loss of cells in the brain. GLD causes severe mental reduction and motoric delay. It is caused by a deficiency in galactocerebroside-β-galactosidase (GALC), which is an essential enzyme in the metabolism of myelin. The disease often affects infants prior to the age of 6 months, but it can also appear during youth or in adults. The symptoms include irritability, fever without any known cause, stiffness in the limbs (hypertony), seizures, problems associated with food intake, vomiting and delayed development of mental and motoric capabilities. Additional symptoms include muscular weakness, spasticity, deafness and blindness.
Currently, there is no curative treatment of GLD. Results from a very small clinical study, including patients with infantile GLD, revealed that children receiving umbilical cord blood stem cells from non-related donors prior to the onset of symptoms, developed with only little neurological invalidity. The results also showed that progression of the disease was stabilized sooner in patients receiving umbilical cord blood as compared to patients receiving bone marrow from adults. It has appeared that bone marrow transplantation has a beneficial effect in patients with mild disease, if performed early in the course of disease. Generally, infantile GLD is lethal prior to the age of 2. Progression of the disease is generally milder in patients with a later onset of the disease.
Exogenous replacement of missing or deficient enzyme (enzyme replacement therapy, ERT) has proven effective in patients with lysosomal storage disorders such as Gaucher disease. Access to an suitable and reliable animal model of GLD will be of outmost importance for the development of enzyme replacement therapy of GLD.
The galactosylceramidase gene (GALC) is about 60 kb in length and consists of 17 exons. Numerous mutations and polymorphisms have been identified in the murine and human GALC gene, causing GLD with different degrees of severity. Table 1 identifies a number of these mutations and polymorphisms, including the very common, so-called 30-kb deletion. This deletion accounts for a major part of the mutant GALC alleles in individuals of European ancestry. This large deletion results in the classic infantile form when in the homozygous state or when heterozygous with another mutation associated with severe disease. Often, however, the observed phenotypic differences in human patients result not only from the particular mutations and polymorphisms, but also from other, yet unknown factors. This makes it almost impossible to predict the effect of each individual mutation if present in a different context.
TABLE 1(adopted from Tappino et al., 2010):Nucleotide (amino acid) substitutionType of mutation127G > C (G43R)Missense188G > A (R63H)Missense3340G > A (E114K)Missense512A > T (D117V)Missense701T > C (I234T)Missense809G > A (G270D)Missense836A > C (N279T)Missense870C > T (S287F)Missense893A > G (Y298C)Missense1027_1036delAAGACAGTTG (K343AfsX3)FrameshiftIVS10del30kbDeletion1138C > T (R380W)Missense1139G > T (R380L)Missense1538C > T (T513M)Missense1609G > A (G537R)Missense1652A > C (Y551S)Missense1739delT (F580SfsX16)Frameshift1853delT (L618X)Nonsense
A natural model of GLD, known as the “twitcher” (twi) mouse, has a mutation of the GALC gene causing complete lack of GALC activity. Various treatments have been attempted on this mouse model, with varying degrees of success. However, the aggressivity of GLD in the twitcher mouse makes this model sub-optimal, since a moderate therapeutic benefit may be superimposed by rapid deterioration. Utility of the “twitcher” mice is further compromised by the fact that the mice typically die at an age of approximately 40 days, the short lifespan resulting in a very small therapeutic window. Moreover, the “twitcher” mouse model has the disadvantage of not being immunotolerant to exogenous, human enzyme. Indeed, the animals may develop a progressive immunological response to repeatedly injected human GALC, which may reduce therapeutic efficacy and/or induce lethal anaphylactic reactions.
Transgenic introduction of human GALC into “twitcher” mice and studies on the transgenic mice have led to the conclusion that low, even undetectable, levels of GALC activity were able to slow the course of GLD in mice. Based on these observations it was predicted that as little as 5% of normal GALC activity would be enough to delay or even prevent symptoms (Gasperi et al., 2004).
Transgenic mice have also been created by homologous recombination, containing a polymorphic change found in humans: The amino acid at codon 168 in murine GALC was changed from histidine to cysteine (R168C). Studies in relation to these mice confirmed that the problem of generating an exact model a human disease by replacing an amino acid in a protein from a mouse or other species is difficult. First of all, transfection studies in COS-1 cells unexpectedly showed that there is little correlation between the effects on GALC activity of particular amino acid substitutions in human GALC and the effects of changing the same amino acids in murine GALC. In particular, while R168C was known to have little effect in humans it had a profound effect on mouse GALC activity. Secondly, the studies also revealed that even though the transgenic mice had considerable, residual expression of GALC, this was able to slow progression of GLD only slightly (Luzi et al., 2002.
Larger animal models of GLD include rhesus monkeys, cats and dogs. However, like the twitcher mice, these natural models of GLD are not immunotolerant to human GALC.
Hence, there is a need for an improved animal model of GLD, which is immunotolerant to human GALC and has a sufficiently slow disease progression and longer lifespan.